Neurotransmitters in carotid body function: the case of dopamine--invited article.

The carotid body (CB) is the main peripheral chemoreceptor. The present model of CB chemoreception states that glomus (type I) cells are the primary receptors, which are synaptically connected to the nerve terminals of the petrosal ganglion neurons. In response to hypoxia, hypercapnia and acidosis, glomus cells release one (or more) transmitter(s) which, acting on the nerve terminals of chemosensory neurons, increases the afferent discharge. Among several molecules present in glomus cells, dopamine, acetylcholine and 5'-adenosine-triphosphate have been proposed to be the excitatory transmitters in the CB. Beside these putative excitatory transmitters, other molecules modulate the chemosensory process through direct actions on glomus cells and/or by producing tonic effects on CB blood vessels. In this review, we focus on the role played by dopamine in the CB chemoreception, with emphasis on the open question if the reported differences on its actions on the generation of afferent chemosensory activity reflect true species differences. The available data suggest that dopamine may play a modulatory role within the cat CB, while in the rabbit CB, dopamine is an excitatory transmitter. Therefore, the reported differences on the actions of exogenously applied dopamine and its participation on the generation of afferent chemosensory activity appear to reflect true species differences.

Evidence for histamine as a new modulator of carotid body chemoreception.

It has been proposed that histamine is an excitatory transmitter between the glomus cells of the carotid body (CB) and the nerve endings of the petrosal ganglion (PG) neurons. The histamine biosynthetic pathway and the presence of histamine H1, H2 and H3 receptors have been reported in the CB. Thus, histamine meets some of the criteria to be regarded as a transmitter. However, there is no evidence that glomus cells contain histamine, or whether its application produces chemosensory excitation. Therefore, we studied its immunocytochemical localization on cat CB and its effects on chemosensory activity. Using perfused and superfused in vitro CB and PG preparations, we assessed the effects of histamine hydrochloride on chemosensory discharges and of histamine H1, H2 and H3 receptor blockers. We found the presence of histamine immunoreactivity in dense-core vesicles in glomus cells. In an in vitro CB preparation we performed pharmacological experiments to characterize histamine effects. The application of histamine hydrochloride (0.5-1,000 microg) to the CB produces a dose-dependent increase in the carotid sinus nerve activity. The H1 receptor blockade with pyrilamine 500 nM produces partial decrease of the histamine-induced response, whereas the H2 receptor blockade (ranitidine 100microM) fail to abolish the histamine excitatory effects. Antagonism of the H3 receptor results in an increase in carotid body chemosensory activity. On the other hand, application of histamine to the isolated PG had no effect on the carotid nerve discharge. Our results suggest that histamine is a modulator of the carotid body chemoreception through H1 and H3 receptor activation.

Cardioventilatory acclimatization induced by chronic intermittent hypoxia.

It has been proposed that chronic intermittent hypoxia (CIH) contributes to generate hypertension in patients with obstructive sleep apnea syndrome and animal models, due to an enhanced sympathetic outflow. A possible contributing mechanism to the CIH-induced hypertension is a potentiation of carotid body (CB) chemosensory responses to hypoxia, but early changes that precede the CIH-induced hypertension are not completely known. Since the variability of heart rate (HRV) has been used as an index of autonomic influences on cardiovascular system, we studied the effects of short and long-term CIH exposure on HRV in animals with or without hypertension. In cats exposed to CIH (PO(2) approximately 75 Torr, 10 times/hr during 8 hr) for 4 days, the ventilatory response to acute hypoxia was potentiated, the arterial pressure remained unchanged, but the HRV power spectrum showed a shift towards the low frequency band. Exposure of rats to CIH (PO(2) approximately 37.5 Torr, 12 times/hr during 8 hr) for 12 days enhanced the ventilatory response to acute hypoxia, but did not increase the arterial pressure. After 21 days of CIH, we found a significant increase of arterial pressure and a shift of the HRV power spectrum towards the low frequency band. Thus, our results support the idea that hypertension induced by long-term CIH was preceded by alterations in the autonomic balance of HRV, associated with an enhance CB chemoreflex sensitivity to hypoxia. Therefore, few days of CIH are enough to enhance the CB reactivity to hypoxia, which contribute to the augmented ventilatory response to hypoxia, and to the early alterations in the autonomic balance of HRV.

Nerve branching and terminal arborizations in the carotid body of the cat. A light microscopic study following anterograde injury filling of carotid nerve axons with horseradish peroxidase.

The terminal arborizations of carotid nerve axons within the carotid body of the cat were densely filled with horseradish peroxidase and studied under the light microscope. Two types of terminal arborizations were found in contact with glomus (type I) cells. The axons differed principally in the wealth of terminal swellings. The largest and most numerous type of arborization consisted of one to several clusters of terminals of variable size and shape arising from a single fiber and distributed in a rather ellipsoidal domain of about 9,000 microns 3 for each cluster. Thus, these arborizations might be in close relation with 20-60 glomus cells. The second type of arborization had substantially fewer terminal swellings, occupying a smaller volume and probably contacted significantly less glomus cells. Both kinds of axons had small rounded and large calyciform endings. The larger arborizations were derived consistently from larger fibers than those which produced the smaller arborizations. The results suggest that the carotid nerve axons generate two types of arborizations within the carotid body. Thus, glomus cells potentially can contact two classes of afferent fibers. The functional significance of a dual chemoreceptor innervation of the carotid body is discussed.

Effects of CO2-HCO3- on catecholamine efflux from cat carotid body.

Using a chronoamperometric technique with carbon-fiber microelectrodes and neural recordings, we simultaneously measured the effects of the following procedures on catecholamine efflux (delta CA) and frequency of chemosensory discharges (fx) from superfused cat carotid body: 1) the addition of CO2-HCO3- to Tyrode solution previously buffered with N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid, maintaining pH at 7.40; 2) hypercapnia (10% CO2, pH 7.10); 3) hypoxia (PO2 h approximately 40 Torr) with and without CO2-HCO3-; and 4) the impact of several boluses of dopamine (DA; 10-100 micrograms) on hypoxic and hypercapnic challenges. With CO2-HCO3-, hypoxia increased fx which preceded delta CA increases, whereas hypercapnia raised fx but did not consistently increase delta CA. Repeated stimuli induced similar fx increases, but attenuated delta CA. After DA, hypoxia produced larger delta CA, which preceded chemosensory responses. Without CO2-HCO3-, hypoxia produced a similar pattern of delta CA and fx responses. Switching to Tyrode solution with CO2-HCO3- at pH 7.40 raised fx but did not increase delta CA. With CO2-HCO3- and after DA, hypoxic-induced delta CAs were larger than in its absence. Results suggest that DA release is not essential for chemosensory excitation.

Electrophysiological evidence for the reconstitution of chemosensory units in co-cultures of carotid body and nodose ganglion neurons.

The electrophysiological characteristics of nodose ganglion sensory neurons, cultured alone or co-cultured with carotid body tissue, were compared. Some properties of the neurons and their response to acid (a carotid body 'natural' stimulus) changed in the presence of this tissue. (a) The evoked action potential after-hyperpolarization was smaller and longer whereas spike amplitude and duration, and the passive membrane properties remained unaltered. (b) Spontaneously occurring action potentials happened more frequently (16% vs 3%). (c) Acid solutions induced appreciable depolarization, an increased discharge, or both, only in a population of co-cultured neurons. These changes probably arose because of synaptic and/or trophic interactions between neurons and glomus cells.

Thermal dependence of chemosensory activity in the carotid body superfused in vitro.

We studied the relationship between chemosensory activity and temperature in carotid bodies excised from pentobarbitone-anesthetized cats, and superfused in vitro at flows between 0.4 and 2.0 ml/min with modified Tyrode's solution buffered with HEPES at pH 7.43. The basal frequencies of chemosensory discharges were recorded from the entire carotid nerve at different steady thermal conditions. For preparations superfused with saline equilibrated with 100% O2, thermally dependent increases in frequency were observed, with significant differences between all nearby thermal stages separated by 0.5 degrees C steps between 36.0 and 38.5 degrees C. The larger gains were recorded between higher temperatures at high flows, between mid temperatures at intermediate flows, and between lower temperatures at low flows. The critical temperature for the calculated maximal gain was directly correlated to superfusion flow. The basal frequencies were consistently elevated when switching to saline equilibrated with 20% O2 and no significant differences in mean ranks were recorded between 36 and 37 degrees C, as between 38 and 39 degrees C, but frequencies at 36-37 degrees C were significantly higher than those at 38-39 degrees C. Brief rises in chemosensory discharges were evoked by injections of NaCN applied to carotid bodies superfused with saline equilibrated with 100% O2. The least effective dose was lower at 40 degrees C than at 37.5 degrees or 35.0 degrees C, but the reactivity and slope were not significantly different. It is concluded that the carotid body chemoreceptors fulfill the criteria for being considered as thermosensors, and that their frequency of discharges is thermally modulated within a range close to physiological body temperature.

Sodium nitroprusside blocks the cat carotid chemosensory inhibition induced by dopamine, but not that by hyperoxia.

We studied the effects of the nitric oxide (NO) synthase inhibitor, Nomega-nitro-L-arginine methyl ester (L-NAME), and the NO donor, sodium nitroprusside (SNP) on cat chemosensory responses to intravenous injections of NaCN (0.1-100 microg/kg) and dopamine (0. 1-20 microg/kg), and to hyperoxic ventilation (100% O2, 60-120 s). Cats were anesthetized with sodium pentobarbitone, paralyzed and artificially ventilated to prevent secondary ventilatory effects. The frequency of chemosensory discharges (fx) was recorded from one sectioned carotid sinus nerve. L-NAME (50 mg/kg i.v.) increased basal fx and slightly potentiated the responses to NaCN and dopamine. SNP (1-2 mg/kg i.v.) increased basal fx, but reduced the NaCN-induced increases of fx over baseline and the transient fx inhibitions induced by dopamine, but not those produced by hyperoxia. Present results indicate that besides the known inhibitory effect of NO on chemosensory responses to low PO2, NO also blocks the chemosensory response to dopamine, leaving hyperoxic responses largely unchanged.

Flow-dependent chemosensory activity in the carotid body superfused in vitro.

The relationship between carotid body chemoreceptor activity and flow was studied in preparations superfused in vitro. The carotid bodies were excised from pentobarbitone-anesthetized cats and superfused with modified Tyrode's solution, buffered with HEPES-NaOH to pH 7.41. The bath temperature was kept constant at 37.7 degrees C. The frequency of chemosensory discharges from the entire carotid nerve was determined during steady-state superfusion with 100% or 20% O2-equilibrated saline at flow rates between 0.15 and 2.95 ml/min, and during 5 min flow interruptions. The peak frequency evoked by flow interruptions was maximal and independent of previous superfusion flows, but the half-excitation time of chemosensory responses to flow interruption was minimal when preceded by superfusion with 100% O2-equilibrated saline at 0.7 ml/min. In steady-state conditions, mean chemosensory activity was higher at lower rates of flow, and, at constant flow, higher under 20% O2 than under 100% O2. To allow comparisons of all data, basal frequencies at given basal flows were referred to their own maximal frequencies evoked by flow interruptions. The best fitting for the relation between basal chemosensory activity and superfusion flow was provided by inverse sigmoid (logistic decay) curves: r = -0.90 and -0.84, at 100% and 20% O2 levels, respectively. The maximal gains were at about 0.78 and 0.86 ml/min, respectively. It is concluded that the chemosensory discharge frequency recorded from carotid bodies superfused in vitro is determined by the superfusion flow, when all other natural chemoreceptor stimuli are held constant.

Lack of correlation between cholinergic-induced changes in chemosensory activity and dopamine release from the cat carotid body in vitro.

We studied the effects of nicotine, acetylcholine (ACh) and dopamine (DA) on the frequency of chemosensory discharges (f(x)) and catecholamine (CA) efflux in the cat carotid body superfused in vitro. CA efflux was measured by changes in CA concentration (DeltaCA) determined by chronoamperometry with nafionated carbon-fiber microelectrodes inserted in the carotid body, while f(x) was recorded simultaneously from the carotid (sinus) nerve. Nicotine (10-20 microg) and ACh (>100 microg) increased f(x) in all carotid bodies (n=16), but produced a delayed DeltaCA ( approximately 0.65 microM) in only half of them. Eserine potentiated ACh-evoked increases in f(x) and CA effluxes. Nicotine and ACh-induced DeltaCA were rapidly reduced upon repeated administration. While f(x) increases evoked by low doses of nicotine or ACh were reduced or abolished by prior administration of exogenous DA (>100 microg), CA effluxes were enhanced and hastened. Thus, cholinergic-induced changes in f(x) are dissociated from CA efflux.

Acetylcholine sensitivity in sensory neurons dissociated from the cat petrosal ganglion.

The petrosal ganglia contain the somata of the sensory fibers of the glossopharyngeal nerves, innervating structures of the tongue, pharynx, carotid sinus and carotid body. Petrosal ganglia were excised from adult cats and their neurons were dissociated and kept in tissue culture for 7-12 days. Intracellular recordings were obtained through conventional microelectrodes. In response to depolarizing pulses, most cells (41/60) presented a 'hump' in the falling phase of their action potentials (H-type), while the remaining neurons lack such hump (F-type). The two types of cells had no differences in resting membrane potential or action potential amplitude. Acetylcholine (ACh) applied locally elicited responses in nearly two thirds of both H-type and F-type neurons tested. Most H-type neurons (17/19) responded with a slow long lasting depolarization, while the remaining (2) did so by generating spikes. In contrast, half of F-type neurons (6/12) responded with one or more spikes and the other half only with a slow depolarization. These results indicate that ACh receptors are present in the soma of many petrosal ganglion neurons subjected to tissue culture, thus supporting the idea that - under normal conditions - their peripheral sensory processes may be excited by ACh.

The chick chorioallantoic membrane promotes survival of co-transplanted rat carotid bodies and nodose ganglia.

Carotid bodies and nodose ganglia, removed from adult rats, were co-implanted onto the chorioallantois of 6- to 12-day chick embryos. Implants were rapidly vascularized and incorporated into the chorioallantoic membrane, where they survived and grew for up to 12 days. The morphological characteristics of grafted tissues were largely preserved. Regenerating axons from nodose neurons invaded the carotid body and contacted some glomus cells through morphologically immature synapses. Thus, the chick chorioallantoic membrane may be a useful substrate to study carotid chemoreceptor-sensory neuron interactions.

Dopamine modulates carotid nerve responses induced by acetylcholine on the cat petrosal ganglion in vitro.

We have recently reported that application of acetylcholine (ACh) or nicotine to the petrosal ganglion-the sensory ganglion of the glossopharyngeal nerve-elicits a burst of discharges in the carotid nerve branch, innervating the carotid body and sinus, but not in the glossopharyngeal branch, innervating the tongue and pharynx. Thus, the perikarya of sensory neurons for the carotid bifurcation exhibit selective cholinosensitivity. Since dopamine (DA) modulates carotid nerve chemosensory activity, we searched for the presence of DA sensitivity at the perikarya of these neurons in the cat petrosal ganglion superfused in vitro. Applications of DA in doses of up to 5 mg to the ganglion did not modify the rate of spontaneous discharges in the carotid nerve. However, if DA was applied 30 s before ACh injections, ACh-evoked reactions were modified: low doses of DA enhanced the subsequent responses to ACh, while high doses of DA depressed the responses to ACh. This depressant effect of DA on ACh responses was partially antagonized by adding spiroperone to the superfusate. Our results show that the response to ACh of petrosal ganglion neurons projecting through the carotid nerve is modulated by DA acting on D(2) receptors located in the somata of these neurons. Thus, dopaminergic modulation of cholinosensitivity could be shared also by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.

Selective activation of carotid nerve fibers by acetylcholine applied to the cat petrosal ganglion in vitro.

The petrosal ganglion innervates carotid body chemoreceptors through the carotid (sinus) nerve. These primary sensory neurons are activated by transmitters released from receptor (glomus) cells, acetylcholine (ACh) having been proposed as one of the transmitters involved in this process. Since the perikarya of primary sensory neurons share several properties with peripheral sensory endings, we studied the electrical responses of the carotid nerve and glossopharyngeal branch to ACh locally applied to the cat petrosal ganglion superfused in vitro. Ganglionar applications of AChCl (1 microg-1 mg) generated bursts of action potentials conducted along the carotid nerve, while only a few spikes were exceptionally recorded from the glossopharyngeal branch in response to the largest doses. Carotid nerve responses to ACh were dose-dependent, the higher doses inducing transient desensitization. Application of nicotine to the petrosal ganglion also evoked dose-dependent excitatory responses in the carotid nerve. Responses to ACh were reversibly antagonized by adding hexamethonium to the superfusate, more intense and prolonged block of ACh responses being produced by mecamylamine. Ganglionar applications of gamma-amino butyric acid and serotonin, in doses of up to 5 mg, did not induce firing of action potentials in any of the branches of the glossopharyngeal nerve. Our results indicate that petrosal ganglion neurons projecting through the carotid nerve are selectively activated by ACh acting on nicotinic ACh receptors located in the somata of these neurons. Thus, cholinosensitivity would be shared by the membranes of peripheral endings and perikarya of primary sensory neurons involved in arterial chemoreception.

Responses to hypoxia of petrosal ganglia in vitro.

NaCN is a classical stimulus used to elicit discharges from carotid body chemoreceptors. The effect is assumed to be mediated by glomus (type I) cells, which release an excitatory transmitter for the excitation of carotid nerve endings. Since the sensory perikarya of the glossopharyngeal nerve (from which the carotid nerve branches) are located in the petrosal ganglion, we tested whether application of this drug to the petrosal ganglion superfused in vitro elicits antidromic discharges in the carotid nerve. NaCN did indeed cause an intense and prolonged burst of nerve impulses in the carotid nerve, while provoking a less intense and much briefer burst of discharges in the glossopharyngeal branch. Carotid nerve responses to NaCN were reduced and shortened by prior or following application of dopamine to the ganglion. Sodium azide applied to the petrosal ganglion evoked a less intense and much briefer burst of impulses in the carotid nerve. Ganglionar application of 2,4-dinitrophenol did not induce discharges in the carotid nerve. Switching the superfusion of the ganglion from a normoxic to a hypoxic solution did not evoke discharges in the carotid nerve. Therefore, the perikarya of carotid nerve neurons are sensitive to NaCN, but are not excited by reducing the pO(2) of the superfusing solution.

Modulatory effect of nitric oxide on acetylcholine-induced activation of cat petrosal ganglion neurons in vitro.

The inhibitory effect of nitric oxide (NO) on carotid chemosensory responses to hypoxia has been attributed in part to an antidromic inhibition of chemoreceptor cells activity. However, NO may also modulate the activity of the primary sensory neurons because NO is produced in the soma of these neurons located in the petrosal ganglion. Since a population of petrosal neurons is selectively activated by acetylcholine (ACh), we studied the effects of NO-donor, sodium nitroprusside (SNP), and the NO-synthase inhibitor, Nomega-nitro-l-arginine methyl ester (l-NAME), on the responses evoked in the carotid sinus nerve (CSN) by ACh applied to the petrosal ganglion in vitro. ACh (1 microgram-1 mg) increased the frequency of action potentials recorded from the CSN in a dose-dependent manner. SNP (10-50 microM) reduced the sensibility and amplitude of the CSN response to ACh, although the maximal response appears less affected. The withdrawal of SNP from the superfusion medium increased the sensibility of the responses to ACh. l-NAME (1-2 mM) slightly increased the sensibility of the ACh-induced responses, effect that persisted after l-NAME withdrawal. These results suggest that NO may play a role as modulator in this autonomic primary sensory ganglion.

Cat carotid body chemosensory responses to non-hypoxic stimuli are inhibited by sodium nitroprusside in situ and in vitro.

We studied the effects of sodium nitroprusside, a nitric oxide donor, on the chemosensory responses to cyanide and nicotine in the cat carotid body. In situ, sodium nitroprusside infusion reduced the cyanide-evoked responses in a dose-dependent manner. In vitro, Tyrode containing nitroprusside reversibly reduced the cyanide- (by 59%) and nicotine-induced (by 45%) chemosensory responses. The present results suggest that chemosensory responses induced by cyanide and nicotine are reduced by increased nitric oxide content, similarly to the hypoxic chemosensory responses.

Adenosine triphosphate-induced peripheral nerve discharges generated from the cat petrosal ganglion in vitro.

Since nucleotides have been postulated as transmitters between glomus cells and chemosensory nerve endings in the carotid body, we studied the effects of their application to the petrosal ganglion, where the perikarya of carotid (sinus) nerve are located. Cat petrosal ganglia were superfused in vitro, while electrical activities of their peripheral processes (carotid nerve and glossopharyngeal branch) were recorded simultaneously. Adenosine triphosphate (ATP) evoked dose-dependent bursts of impulses in carotid nerve, while those in glossopharyngeal branch were less intense and consistent. Adenosine monophosphate was less effective than ATP. ATP-induced carotid nerve responses presented no temporal desensitization and persisted after applying P(2Y) receptor blocker Reactive Blue 2 to the ganglion. The results indicate that ATP has an excitatory effect on the perikarya of the population of petrosal ganglion neurons projecting peripherally through the carotid nerve.

Fast activity and oscillatory potential of carp retina in the frequency domain.

There are two kinds of fast activity in the ERG: fast retinal potentials (FRP), an irregular series of spiky wavelets and oscillatory potentials (OP), a rhythmic sequence of events. Corneal ERG from nine intact young carps, evoked by extended pulses of diffuse white light under mesopic adaptation, displayed two different groups of wavelets related to ON and OFF, respectively. Stimulation and recording conditions were established to permit separate Fourier analysis of both groups of wavelets. Power distributions of normalized ON spectra showed both a wide dispersion and a high inter-subject variability. All normalized OFF spectra showed, instead, components within a narrow band from 52 to 56 Hz, most of them maximum relative power peaks. It is concluded that FRP originating in highly labile sources dominate ON fast activity, while the predominant OFF fast activity are OP originating in a stable discrete source.

CONFREG: a BASIC program for calculating and plotting confidence regions based on correlational analyses.

Many observations encountered in biological and medical research are randomly distributed in bivariate scales, and thus not susceptible to simple regression analyses. Since such data are depicted by ellipses in scatter diagrams, a computer program to calculate the confidence regions for the means or the total data of bivariate samples was written in BASIC for correlational analyses. The program, based on the principal axes algorithm, plots the calculated confidence regions as an elliptic area, using the fitted equations for its major and minor axes. The program displays the sample parameters required to perform comparisons between different groups of experimental conditions.